Expression of galectin-3 by exudated neutrophils drives neutrophil apoptosis and clearance in a model of self-resolving peritonitis. Galectin-3 has been associated with a plethora of proinflammatory functions because of its ability, among others, to promote neutrophil activation and because of the reduction in neutrophil recruitment in models of infection in Gal-3-null mice. Conversely, it has also been linked to resolution of inflammation through its actions as an opsonin and its ability to promote efferocytosis of apoptotic neutrophils. Using a self-resolving model of peritonitis, we have addressed the modulation and role of Gal-3 in acute inflammation. We have shown that Gal-3 expression is increased in neutrophils that travel to the inflamed peritoneum and that cellular localization of this lectin is modulated during the course of the inflammatory response. Furthermore, neutrophil recruitment to the inflamed peritoneum is increased in Gal-3–null mice during the course of the response, and that correlates with reduced numbers of monocytes/macrophages in the cavities of those mice, as well as reduced apoptosis and efferocytosis of Gal-3–null neutrophils. These data indicate a role for endogenous Gal-3 in neutrophil clearance during acute inflammation. N-acetyllactosamine residues on cells [Streptococcus pneumoniae, Gal-3 appears to be beneficial for the host, with enhanced pathogenicity observed in Gal-3–null mice and reduced infection in mice treated with recombinant Gal-3 [S. pneumoniae, and that can be rescued by administration of the recombinant protein [Escherichia coli or to air pouches inoculated with the Leishmania major substrain Friedlin when compared with substrain LV39 [S. pneumoniae and LV39 induced release of Gal-3 into the inflammatory exudate, whereas E. coli and Friedlin did not, suggesting that it is the presence of Gal-3 in inflammatory exudates that functions to drive neutrophil recruitment. This hypothesis is supported by the finding that administration of recombinant Gal-3 into the murine air pouch results in neutrophil recruitment [Galectins are a family of β-galactoside–binding proteins that elicit their effects by binding to exposed on cells . Severalon cells –5. Durinnt Gal-3 , 7. Suchnt Gal-3 . Adminisnt Gal-3 . Decreas protein ; Gal-3 cain LV39 . Interesruitment . Whetherruitment  observedruitment  observedruitment .As well as the aforementioned studies supporting a role for Gal-3 in driving innate immunity, there is evidence to suggest that it may facilitate resolution of inflammation through its actions as an opsonin ; furtherS. pneumoniae–infected alveoli. Human neutrophils express Gal-3, and levels are not modulated upon migration through an endothelial monolayer in an in vitro transmigration assay in response to ILN-8 [Although the studies mentioned provided evidence that Gal-3 is an important regulator of the innate immune system, the modulation of the endogenous protein in innate immune cells during the course of an inflammatory response has not, to our knowledge, been studied systematically. The expression of Gal-3 is reportedly negligible basally in both human and murine neutrophils, and whether levels are modulated during inflammation is not clear because, again, contrasting reports exist. Gil et al.  demonstrto ILN-8 .In this study, we characterized the regulation and function of Gal-3 in neutrophils during the course of a self-resolving inflammatory response. Our findings demonstrate that Gal-3 expression is increased in neutrophils that have migrated into the inflamed peritoneal cavity and that neutrophil numbers are increased in the peritoneal cavities of Gal-3–null mice. This is likely due to reduced clearance rather than increased trafficking, as evidenced by reduced levels of apoptosis in Gal-3–null neutrophils coupled with their reduced efferocytosis.FITC apoptosis detection kit was purchased from BD Biosciences . CellTrace carboxyfluorescein succinimidyl ester was purchased from Thermo Fisher Scientific . Histopaque 1077 and Zymosan were purchased from Sigma-Aldrich .All Abs were purchased from eBioscience . The Annexin VAll animal studies were conducted with ethical approval from the Queen Mary University of London Local Ethical Review Committee and in accordance with the United Kingdom Home Office regulations .http://functionalglycomics.org), and a colony was established in Margate, United Kingdom, at the Charles River Laboratories . These mice were on a C57BL/6 background, and age- and sex-matched controls were used for all experimental work. All animals were fed standard laboratory chow and water ad libitum and were maintained on a 12-h light–dark cycle under specific pathogen-free conditions. All experiments were performed with mice 6–7 wk old.Male C57BL/6 mice were obtained from Charles River . Breeding pairs of Gal-3–null mice  were provided by the Consortium for Functional Glycomics  i.p. in 1 ml sterile PBS, as previously described by Ajuebor et al.  in 1999;E. coli  i.p. in 1 ml sterile PBS. Peritoneal cavities were lavaged, as described above, at 4 h postinjection.In some experiments peritonitis was induced using 1 mg lyophilized Murine cells were incubated with Abs for Ly6G  to identify neutrophils or a combination of F4/80 and Gr-1  to identify classic and nonclassic monocyte/macrophages. Cells were then fixed and permeabilized with BD fixation and permeabilization buffer before the addition of the anti–Gal-3 Ab  to assess intracellular Gal-3 levels . To determine cytosolic expression of Gal-3, mean fluorescence intensities for surface expression were deducted from the mean fluorescence intensity values obtained for total expression. In all cases, Abs or relevant isotype controls were incubated at 4°C before flow cytometric analysis using FlowJo software .Peritoneal lavage cells were seeded onto Ibidi 8-well chamber slides in RPMI 1640 and allowed to adhere. After fixation with 1% paraformaldehyde, cells were blocked with 5% FBS before permeabilization with 0.05% Triton X-100. Staining for intracellular Gal-3 was performed using anti–Gal-3–PE (eBioscience) for 1 h at room temperature. Cells were washed 3 times and then incubated with phalloidin 647 for 20 min at room temperature. Finally, cells were mounted with ProLong Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific). Immunofluorescence was assessed using a Zeiss LSM 710 confocal microscope . Images were processed using Zen 2012 (Carl Zeiss Microscopy) and Adobe Photoshop CS6  software.6/ml; 5 µM CFSE (Thermo Fisher Scientific) was added, and cells were incubated at 37°C for 8 min. An equal volume of FCS was then added to quench excess CFSE, and the cells were washed. After 2 additional washes with ice-cold RPMI 1640, the cells were resuspended in ice-cold PBS at a density of 25 × 106/ml. Then, 5 × 106 neutrophils were administered to each Gal-3–null recipient mouse via the lateral tail vein, and 1 mg zymosan was injected i.p. 4 h after zymosan administration; peritoneal lavages were collected, as described above, and cells were stained with anti-Ly6G to identify neutrophils and Gal-3. Some cells were also permeabilized after the surface stain with Ly-6G to assess intracellular expression of Gal-3.WT mice were euthanized, and tibias and femurs were collected, cleaned, and flushed with RPMI 1640  using a 25-guage needle, as described previously . FollowiGal-3 levels were measured in peritoneal exudates using a commercial ELISA kit  according to manufacturer’s instructions.−, Zombie−) and late apoptotic (Annexin V–Zombie double positive) cells were gated, and Gal-3 expression assessed.Exudated neutrophils from WT and Gal-3–null mice were collected after 4, 48, 72 and 96 h of zymosan-induced peritonitis and were labeled with Ly6G. Then, the cells were labeled with Annexin V and propidium iodide as per manufacturer’s instructions (BD Biosciences) or Zombie NIR viability dye  to assess apoptosis. In some experiments, 4-h exudated neutrophils were cultured overnight in RPMI 1640 and assessed the following day at 18 h for surface Gal-3 levels and apoptosis. Gal-3 staining was performed as described above, and apoptosis levels were assessed using Annexin V and Zombie NIR. Viable  for 5 min at 37°C. After washing, neutrophils were resuspended in RPMI 1640 at 2 × 106/ml, and 0.5 ml was added to each well of macrophages and incubated for 1 h. After extensive washing efferocytosis was quantified using ImageJ software . Images were split into their individual red/blue/green channels, and the same background threshold was applied to all images before quantification of fluorescence.Biogel-elicited macrophages were collected from WT mice, as previously described , and wersem of n experiments. All data were tested for normal distribution, and power calculations were performed using G*Power software  [t test for 2 groups, 1-way ANOVA, followed by a Bonferroni or Dunnett’s post hoc test  or 2-way ANOVA, followed by Bonferroni post hoc test. In all cases a P value < 0.05 was considered significant to reject the null hypothesis, and differences were considered significant.Statistical significance was assessed using SPSS computer software . Data are expressed as means ± Germany) . StatistTo investigate the endogenous levels of Gal-3 during the course of an acute inflammatory response, a zymosan-induced peritonitis was performed and monitored for 96 h; by which time, the inflammation had resolved. Because of the discrepancy among reports of Gal-3 expression in neutrophils, we fully investigated expression levels in 3 sources of neutrophils: those from the bone marrow, those from the peripheral blood, and those that had migrated into the inflamed peritoneal cavity. There was a rapid increase in the number of total leukocytes in the peritoneal cavity within 4–24 h after zymosan administration, and that number remained elevated during the course of the response, with increased leukocyte numbers still observed at 96 h compared with basal levels . The numAnalysis of peritoneal lavage fluid by ELISA revealed baseline levels of 2.155 ± 0.450 ng/ml Gal-3 within the cavity. These levels increased significantly during the peritonitis time course, with peak levels at 72 h (7.293 ± 1.239 ng/ml) . EndogenTo determine the cellular localization of Gal-3, flow cytometry was performed on nonpermeabilized vs. permeabilized neutrophils .+ neutrophils were clearly detectable within peritoneal lavages, as shown in To determine whether the intracellular Gal-3 detected was derived from the extracellular environment, that is, being taken up from the inflammatory exudate or being produced by the neutrophils themselves, an adoptive transfer experiment was performed. CFSE-labeled bone marrow neutrophils isolated from WT mice were injected i.v. into Gal-3–null mice, and a zymosan-induced peritonitis performed. Neutrophils within lavage fluid were identified based on their characteristic forward/side light scatter and following doublet exclusion, Ly6G+ neutrophils were identified . Again, Gal-3 was readily detectable within the cytosol of neutrophils that had traveled to the peritoneum in response to both E. coli and zymosan , whereas levels were low on the surface of zymosan-elicited neutrophils as well as on those elicited by E. coli. To investigate whether neutrophils lacking Gal-3 undergo apoptosis at a different rate than cells from WT animals, neutrophils collected from the peritoneal cavity were cultured overnight, and levels of apoptosis were assessed by flow cytometry. Upon removal from the cavity, most neutrophils were viable in both genotypes (Neutrophil recruitment in Gal-3–null mice appears to vary in its magnitude depending on the inciting stimuli and the site of the inflammation. We, therefore, performed a peritonitis experiment in which we used enotypes . Becauseenotypes . Interesenotypes .Because there is evidence in the literature that Gal-3 is involved in neutrophil clearance, a key facet of the resolution process, leukocyte trafficking was compared in Gal-3–null mice and their WT counterparts at 48–96 h after zymosan. At 48 h after zymosan, total leukocyte counts were comparable for WT and Gal-3–null mice; however, at 72 h, there was a trend toward more leukocytes in the peritoneal cavities of Gal-3–null mice, although that increase did not reach statistical significance, and levels had declined to those observed in WT mice by 96 h , indicatThe modulation of the inflammatory process, through effects on immune cell biology, by members of the galectin family is an ever-growing area of research. The focus has, however, been largely on the T cell–driven models of autoimmune disease and, in the case of Gal-3, in cancer. There are several reports describing the effects of Gal-3 on neutrophil activation and its importance in models of infection, in which it might function as an alarmin by augmenting the inflammatory response . With thPrevious studies have failed to detect expression of Gal-3 intracellularly in murine neutrophils, although it is readily detectable on the cell surface , 11. ThiE. coli infection also resulted in a significant enhancement of neutrophil trafficking to the lungs of Gal-3–null mice [E. coli increases neutrophil migration to the lungs in Gal-3–null mice is in contrast to our findings in the peritoneal cavity. This highlights the differences among distinct anatomic sites and might be due to the effects of Gal-3 on stromal cells at different sites. A different strain of mice was used in this study, which may also account for the observed differences. Further similarities exist between these inciting stimuli, which may account for the differing responses seen. Streptococcus pneumoniae infection increases Gal-3 levels in bronchoalveolar lavage fluid as high as 50 μg/ml; likewise, cutaneous infection with L. major LV39 results in increased levels of extracellular Gal-3 and is associated with enhanced neutrophil recruitment. In contrast, infection with E. coli or the L. major substrain Friedlin failed to significantly induce Gal-3 release, and a role for Gal-3–dependent neutrophil recruitment was not observed. Although we observed Gal-3 within the peritoneal exudate and levels were significantly increased during the course of the response, the amount detectable was in the low nanograms per milliliter range, and similar to what was observed after E. coli infection, neutrophil recruitment was increased, rather than decreased, in Gal-3–null mice. Another important factor might be the mechanism by which neutrophils travel to the inflammatory site. Migration in response to zymosan and E. coli is known to be dependent on β2 integrins [S. pneumoniae is independent of β2 integrins, and it is thought that, under these circumstances, Gal-3 is able to act as an adhesion molecule to facilitate neutrophil trafficking, which likely explains the requirement for high levels of the protein extracellularly in these models. Our data suggest that the observed enhancement in neutrophil numbers within the peritoneal cavity of Gal-3–null mice is due to alterations in neutrophil clearance, rather than a direct role on neutrophil trafficking per se, although a direct role cannot be ruled out.Struck by this significant increase in Gal-3 expression upon migration to the inflammatory site, we have sought to address the role of neutrophil Gal-3 through investigation of the profile of neutrophil recruitment, apoptosis, and clearance in the zymosan peritonitis model. Increased levels of neutrophil recruitment into the peritoneal cavity of Gal-3–null mice were observed at 4, 72, and 96 h. This is in contrast to the reported response to thioglycollate in previous studies, as well as in models of pneumonia, in which neutrophil migration to the peritoneal cavity and lungs, respectively, was reduced in Gal-3–null mice , 12. Sucntegrins , which fntegrins . In contIt is important to consider the cellular localization of galectins when studying their actions. Published reports on the effects of Gal-3 have shown cellular localization affects its function in the induction of apoptosis in T cells. Extracellular Gal-3 induces T cell apoptosis , whereasMacrophages are crucial to the resolution process, with essential roles in the clearance of apoptotic neutrophils . PreviouTogether, our data highlight a role for endogenous Gal-3 in the host phagocyte function, enhancing neutrophil apoptosis and clearance. Failure to eliminate dying neutrophils leads to tissue damage and dissemination of cellular contents, which can have major pathologic consequences, particularly in infection.R.D.W performed experiments, analyzed data, and wrote the manuscript. P.R.S., P.T., and M.B.F. performed experiments and analyzed data. L.V.N. performed experiments, analyzed data, and wrote the manuscript, and D.C. designed and performed experiments, analyzed data, and wrote the manuscript.


Now, using a suture-button device to treat distal tibiofibular syndesmotic injuries is overwhelming due to its advantages over screw fixation. Current systematic review was conducted to make a comparison between suture-button fixation and traditionally screw fixation in the treatment of syndesmotic injuries. The outcomes included functional outcomes, implant removal, implant failure, malreduction, post-operative complications , and cost-effectiveness aspects.A literature search in the electronic databases of Medline, Embase, the Cochrane Library, Web of Science was conducted to identify studies until March 2017. The references of the included articles were also checked for potentially relevant studies. Only English articles were included. We followed the Preferred Reporting Items for Systematics reviews and Meta-Analysis (PRISMA) guidelines in this review.Finally, 10 studies were identified, encompassing a total of 390 patients. The mean American Orthopaedic Foot and Ankle Society ankle score (AOFAS) score of 150 patients treated with the suture-button device was 91.06 points, with an average follow-up of 17.58 months, and the mean AOFAS score of 150 patients treated with syndesmotic screws was 87.78 points, with an average follow-up of 17.73 months. Implant removal was reported in 5 of 134 (3.7%) patients treated with the suture-button device, and in 54 of 134 (40.2%) patients treated with the syndesmotic screw. No patient in the suture-button fixation group had implant failure, however the rate of implant failure in the screw fixation group was 30.9%. Malreduction was reported in 1 of 93 (1.0%) patients treated with the suture-button device, and in 12 of 95 (12.6%) patients treated with the syndesmotic screw. The rate of post-operative complications in the suture-button fixation group was 12.0% and 16.4% in the screw fixation group. There was only one publication demonstrated about cost-effectiveness aspects, it showed that patients treated with the suture-button device spent on average $1482 less and had a higher quality of life by 0.058 quality-adjusted life-year compared with patients who received fixation with 2 syndesmotic screws in supination-external rotation type 4 injuries.Based on our research, though the suture-button fixation group had similar functional outcome (measured on the AOFAS score) and post-operative complication rate compared with the syndesmotic screw fixation group, the suture-button device could lead to better objective range of motion (ROM) measurements and earlier return to work. Besides, the suture-button fixation group had lower rate of implant removal, implant failure, and malreduction. However, high-quality randomized controlled trials with more uniformity in outcome reporting are desirable to determine the long-term effects and cost-effectiveness of the suture-button device. Syndesmotic injuries arise in approximately 13% of all patients with ankle fractures which are commonly seen in pronation and external rotation injuries, and in approximately 20% of ankle fractures requiring operative fixation [Though screw fixation as the gold-standard in treatment of syndesmotic injury, some significant issues should be considered, such as screw loosening, breakage, discomfort, reoperation, loss of reduction due to early implant removal –7. More The study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria (PRISMA).To be included in our analysis, the study had to: (1) evaluate a comparison between suture-button fixation and traditionally screw fixation in the treatment of syndesmotic injuries. (2) the studies included at least one of the outcome measures. Studies were excluded: (1) case reports, conference abstracts, and publications that were only discussing screw fixation or suture-button. (2) pediatric or cadaveric studies (3) studies not published in English. (4) duplicate publication.A literature search was conducted to identify studies in which made a comparison between suture-button fixation and traditionally screw fixation in the treatment of distal tibiofibular syndesmotic injuries. The following key terms were combined with Boolean operators in the search: “syndesmo*”, “tibiofibular”, “TightRope”, “suture button”, “screw”, The last search was updated on March 2017. We only included English articles. An additional search of the references of included studies was performed to find relevant studies.The following data were independently extracted from each of the included studies by two investigators (Y Liang and YC Fang): name of the first author, year of publication, targeted population, study type, mean-age, suture-button usage, cortical screw usage, rehabilitation process, follow-up, and the outcomes of the systematic review including: main functional outcomes, implant removal, implant failure, malreduction, post-operative complications , and key findings. When disagreement existed, it was resolved by consulting another investigator (PT Chen).The quality of the randomized controlled trials (RCTs) was assessed according to the Cochrane risk assessment scale, including details of the methods of random sequence generation, allocation concealment, blinding, incomplete outcome data, selective outcome reporting, and other sources of bias. The Methodological Index for Non-Randomized Studies (MINORS) Criteria was used to assess non-RCTs and was scored from 0 to 24 . The assA total of 150 potentially relevant references preliminarily reviewed. By scanning the titles and abstracts, 11 articles that met the inclusion criteria were reviewed for full-text screening. After full texts assessed for eligibility, 1 article  was exclEach included randomized controlled trials , 9, 15 sThe main characteristics of 9 studies were shown in Tables n = 21) with screw fixation (n = 19), predominantly Weber C fractures. A standing cone-beam CT-scan with bone algorithm was performed at final follow-up to qualify the grade of osteoarthritis. No significant differences in functional results between groups were detected at the last follow-up. However, all functional scores were lower in patients who had malreduced syndesmosis on final follow-up than in patients with anatomically reduced syndesmosis. CT evaluation showed a low malreduction rate in both groups and both methods maintained reduction well. The incidence of osteoarthritis showed no significant difference between the two groups.Kortekangas et al.  publishep = 0.45) and dorsal flexion (p = 0.43) at last follow-up, however, the ankle range of motion was higher in the dynamic fixation group at all times when considering the plantar flexion, although this difference was minor for dorsal flexion.; (3). Patients in the TightRope fixation group seemed to be with less pain according to visual analogue scale (VAS); Regarding radiologic results, though adequate reduction was achieved after the surgery in both groups except for 1 patient in the screw group who required a corrective surgery , patients in the screw fixation group had significantly higher loss of reduction (P = 0.0005). Significant loss of reduction  was observed in 4 patients  in the static fixation group. However, the TightRope fixation group showed no significant loss of reduction. The reoperation rate was significantly higher in the static fixation group (P = 0.006) Both groups had 1 patient with partial syndesmotic ossification.Laflamme et al.  conductet = −2.43; P = 0.02).Naqvi et al.  publisheKocadal et al.  performep = 0.01) and at 12 months (p = 0.04) postoperatively and earlier return to work than the screw fixation group . In addition, most of the patients were satisfied with the suture-button device while a greater number of fair or poor results existed in patients who had syndesmosis screw fixation. They concluded that the suture-button device could accelerate rehabilitation and improve outcomes.Thornes et al.  publishep < 0.05). Seventeen patients in the screw fixation group had a second operation for implant removal at an average of 4.38 months.Cottom et al.  conductep = 0.054) and subjectively reported less stiffness and discomfort.Coetzee et al.  publishen = 24) and screw fixation (n = 20). The fracture types were SER IV,SER III,SA I,SA II,PER IV,PER III,PA III, PA II. Regarding tibiofibular clear space, the improvement in the screw fixation group was statistically significant (p = 0.01),but the improvement in the suture-button fixation group was not (p = 0.05). However, the suture-button fixation group fully recovered to a normal ROM. Metal screws had broken in 5 of 24 patients (20.8%). No statistically significant difference was found between the two techniques according to AOFAS scores and VAS scores.Kim et al.  performep < 0.01).Seyhan et al.  performeSeveral different scoring systems were applied to evaluate the functional outcome between the suture-button fixation group and the screw fixation group, such as: AOFAS score, the Olerud–Molander score, modified AOFAS scoring scale, the SF-12 health questionnaire, the Foot and Ankle Disability Index score, and so on. As the AOFAS score was the most used outcome, thus, we selected it to make a comparison. The AOFAS score of 150 patients treated with the suture-button device was 91.06 points, with an average follow-up of 17.58 months. The AOFAS score of 150 patients treated with syndesmotic screws was 87.78 points, with an average study follow-up of 17.73 months.The outcome of implant removal was reported in 7 studies , 17, 18.Seven publications demonstrated implant failure , 15, 17.The outcome of malreduction was reported in 4 studies , 11, 17.Though the implant failure and malreduction are considered as post-operative complications, we have already interpreted them respectively. On current section, we preferred to make a comparison of other complications such as infection, soft-tissue irritation, discomfort, syndesmosis ossification and so on. The outcome of complications was reported in five studies , 15, 17.In our search, only one publication  was idenThough current gold standard to treat syndesmotic injuries is syndesmotic screw fixation, using a suture-button technique has been raised more and more interest and increased rapidly over the last decade. In the current review suture-button fixation group shows similar AOFAS outcome scores (91.06 points) compared to conventional screw fixation (87.78 points) group. The rate of implant removal and malreduction was lower in the suture-button fixation group. Besides, no patient in the suture-button fixation group had implant failure, however the rate of implant failure in the screw fixation group was 30.9%. In addition, the rate of post-operative complications was lower in the suture-button fixation group (12.0% versus 16.4%). There was only one publication demonstrated about cost-effectiveness aspects, it concluded that using a suture-button device is more economical than syndesmotic screws not removed on a routine basis in the treatment of supination-external rotation type 4 injuries.The need for routine syndesmotic screw removal and the time to screws removal are still controversial. All publication except one  in this Many previous investigations evaluating TightRope fixation for syndesmotic injuries have reported 0% malreduction rates, but they used only plain radiography to assess malreduction , 25–28. The main complications reported in the included studies were infection, soft-tissue irritation, discomfort, syndesmosis ossification and so on. In current review, the suture-button fixation group showed similar risk of post-operative complications with the screw fixation group. Regarding the suture-button fixation group, some literatures suggested some modifications in surgical procedure, such as a posterior short knot and/or reaming the posterior aspect of fibula which was useful to reduce the incidence rate of infection, irritation and discomfort , 30. Thep = 0.01) with no patients in the suture-button group required implant removal [The dynamic nature of the suture button device theoretically could allow some degree of physiologic micromovement of the syndesmosis, leading to earlier return to full weight-bearing and better objective ROM measurements. However, screw fixation does not allow normal motion of the syndesmosis during healing because the screw may break or loose. Thornes et al. noted that patients in the suture-button group were kept no weightbearing for a significantly shorter mean time than patients in the syndesmotic screw group . As function outcomes are influenced by severity of trauma, presence of cartilage injury, soft tissue healing, subjective sensation of patients and so on, it is more appropriate to assess syndesmotic injuries based on radiologic parameters rather than functional scores. The key point is the accurate anatomic reduction of the syndesmotic injuries. Thus, more high-quality studies comparing the reduction outcomes of screw fixation and suture-button fixation should focus on radiologic evaluation. 2). Computed tomography as a useful tool for the detection of minor syndesmotic diastasis should be mostly used to make the assessment of postoperative malreduction , 34. Bil1). The publication of Coetzee et al.  was an oBased on our research, though the suture-button fixation group had similar functional outcomes (measured on the AOFAS score) and post-operative complication rate compared with the syndesmotic screw fixation group, the suture-button device could lead to better objective range of motion measurements and earlier return to work. Besides, the suture-button fixation group had lower rate of implant removal, implant failure, and malreduction. However, high-quality randomized controlled trials with more uniformity in outcome reporting are desirable to determine the long-term effects and cost-effectiveness of the suture-button device.


The adsorption of HIB also caused significant peak-shifts (lower frequency shift around 170 cm−1 and higher shift around 186 cm−1) and an intensity change (around 100–150 cm−1 and 270–290 cm−1) in the radial breathing mode of Raman spectra. The charge transfer from SWCNT to HIB was further confirmed by the change in the C1s peak of X-ray photoelectron spectrum, revealing the oxidation of carbon in SWCNT upon HIB adsorption.Electron density of single wall carbon nanotubes (SWCNT) is effectively modified by hexaiodobenzene (HIB) molecules using liquid-phase adsorption. UV-Vis-NIR absorption spectra of the HIB-adsorbed SWCNT, especially in the NIR region, showed a disappearance of S Hayakawa et al. [6I6) molecules exhibit a two-electron oxidation that generates a di-cation (C6I6)2+ from HIB (C6I6) [6I6)2+ shows σ-aromaticity co-existing with the conventional π-aromaticity that it shares with its neutral parent [Since their discovery , single et al. , who obsB (C6I6) . In addil parent . Becausel (46.2 mg L−1), and HIB@SWCNT-h (127.2 mg L−1) were measured under N2 environment as shown in l and 18.6 wt % for HIB@SWCNT-h, respectively. The amount of adsorbed HIB per gram can be calculated by the following equation:−1 for HIB@SWCNT-l and 228.5 mg g−1 for HIB@SWCNT-h.A TGA provides a quantitative way to determine the thermal stability of SWCNT as well as the amount of HIB molecules adsorbed on SWCNT. The TGA curves of HIB, SWCNT, HIB@SWCNT-l, and HIB@SWCNT-h. The UV-Vis-NIR spectrum of SWCNT has the absorption bands in the range of 1200–2100 nm (S11), 700–1100 nm (S22), and 500–700 nm (M11), originating from the interband electronic transitions of van Hove singularities in semiconducting and metallic SWCNT [ic SWCNT .11 transition after the HIB adsorption indicates the charge transfer between HIB and SWCNT. Furthermore, it should be noted that the intensities of these peaks decrease with increasing the adsorbed amount of HIB and they almost disappear with higher adsorption. This effect can be interpreted either as the withdrawal of electrons from the valence band  or as the injection of electrons to the conductance band  in semiconducting SWCNT [11 indicates the weak interaction between metallic SWCNT and adsorbed HIB.The change in Sng SWCNT . Howeverl, HIB@SWCNT-h, and HIB@SWCNT-HTT at the radial breathing mode (RBM) region. In liquid-phase adsorption, the influence of the solvent cannot be neglected. In order to examine the contributions of HIB or THF to the Raman spectrum, the spectrum of SWCNT/THF  is also shown in −1 shifted to the lower frequency side after the adsorption of HIB: The observed shift was by 3.4 cm−1 for HIB@SWCNT-l and 5.8 cm−1 for HIB@SWCNT-h. Further, a significant decrease in the intensities of all the other peaks (from 250 to 300 cm−1 and from 100 to 150 cm−1) was observed: in particular, the peaks at 140 cm−1 disappeared completely. However, SWCNT/THF shows no significant changes in this region. This indicates that the changes in the electronic density of SWCNT can be attributed to the HIB adsorption but not to THF, and it is also further confirmed by HIB@SWCNT-HTT. In the Raman spectrum of HIB@SWCNT-HTT, all peaks are restored to their original state after the removal of HIB molecules from SWCNT by a heat treatment. The disappearance of the RBM peak at 140.0 cm−1, suggests a loss of resonance from the nanotubes with a diameter of 1.74 nm  upon th32/d+6.5 . This re−1  should be caused by a convolution of the peak at 169.8 cm−1 of SWCNT and 166.4 cm−1 of HIB. On the other hand, the peak at 186.4 cm−1 displays a red-shift upon the HIB adsorption, which points to the hardening of radial breathing motion of carbon atoms in SWCNT with a diameter of 1.29 nm [It should be noted that the Raman spectra of HIB-adsorbed SWCNT are shifted both towards higher and lower frequencies. In detail, the small blue-shifts of the peak at 169.8 cm 1.29 nm ,20. The  1.29 nm ,21,22. Fh. Peak fitting involving a combined Lorentzian and Gaussian function was performed to give better understanding on the origins of binding energies  upon the HIB adsorption, evidencing the charge transfer between SWCNT and HIB. The downshift of the binding energy, consistent with the shift in Fermi level toward the valence band edge, can be interpreted as evidence of the decrease in the electron density in the SWCNT with adsorbed HIB, indicating a charge transfer from SWCNT to HIB. A similar behavior was reported by Mistry et al. [et al. [Systematic XPS analyses were performed to understand the direction of charge transfer between SWCNT and HIB molecules. be walls . The othbe walls ,25,26. Ty et al.  for nitr [et al.  for AgX 2 mixed gases for 1 h. For the preparation of HIB-adsorbed SWCNT, a typical procedure was as follows: 3 mg of SWCNT were dispersed in 50 mL tetrahydrofuran (THF) solution by ultrasonication using an ultrasonic cleaner  at 298 K for 2 days. Afterwards, hexaiodobenzene (HIB) (2.31 mg and 6.36 mg) was added to the SWCNT dispersion, and then the mixture was further dispersed by sonication for 15 min. The samples were denoted as HIB@SWCNT-l and HIB@SWCNT-h, respectively.Super-growth SWCNT were synthesized by a chemical vapor deposition (CVD) process . The SWCNT samples were used without further purification because of their high carbon purity (higher than 99.98%) . Tube cah sample was further heat-treated up to 1273 K at a ramp rate of 5 K min−1 under N2 at a flow rate of 100 cm3 min−1. This sample was denoted as HIB@SWCNT-HTT.Then, the mixture was moved into a water bath, and kept at the temperature of 298 K for 1 week to reach the adsorption equilibrium. After filtration, the remaining solid was washed with THF to remove the free HIB molecules and was dried under vacuum at 373 K overnight. After the analysis, a HIB@SWCNT-−1 and N2 flow rate of 100 cm3 min−1. The changes in the electronic properties of SWCNT upon HIB adsorption were measured through the following methods. Raman spectra of each sample were measured on a dried solid by Raman spectrometer  with the excitation wavelengths of 532 nm (power 0.1 mW). X-ray photoelectron spectra (XPS) were measured with X-ray photoelectron spectrometer  using monochromatized MgKα radiation as a photon source. The optical absorption spectra measurement  was performed on an HIB-adsorbed SWCNT solution which was prepared by dispersing HIB-adsorbed SWCNT in THF (20 mg L−1) by ultrasonicated for 24 h. High-resolution transmission electron microscope  observation was carried out by drop-casting dispersed solution of HIB-adsorbed SWCNT onto carbon-film-supported copper grids.Thermogravimetric analysis (TGA) was performed on a thermogravimetric analyzer  at a heating rate of 5 K min−1 and an up-shift in the RBM-band at 186.4 cm−1 provide evidence for the effective charge transfer from SWCNT to HIB. The slight downshift of C1s XPS peak revealed a slight oxidation of carbon in SWCNT upon HIB adsorption. From these results, we conclude that HIB act as an electron acceptor for SWCNT.By using liquid-phase molecular adsorption of HIB to SWCNT, the electronic structure of SWCNT could be modified. The electronic structure changes of SWCNT were investigated by spectroscopic methods. UV-Vis-NIR absorption spectra analysis showed that the interaction of HIB could induce a change in the electron density of state in SWCNT. The disappearance of the RBM-peak at 140 cm


As a control, Ni-Ti orthodontic wire, diameter 0.016 in, was used. The three-point bending properties were evaluated in a modified three-point bending system for orthodontics. The static friction between the orthodontic wire and the bracket was also measured. The load-deflection curves were similar among Ni-Ti and PEEK wires, except for 16PEEK with slot-lid ligation. The bending force of 19-25PEEK wire was comparable with that of Ni-Ti wire. 19-25PEEK showed the highest load at the deflection of 1500 μm (p < 0.05) in the case of slot-lid ligation. No significant differences were seen in the permanent deformation between Ni-Ti and all three PEEK wires (p > 0.05). No significant difference was seen in static friction between all three PEEK wires and Ni-Ti wire (p > 0.05). It is suggested that 19-25PEEK will be applicable for orthodontic treatment with the use of slot-lid ligation.Polyetheretherketone (PEEK) is now attracting attention as an alternative to metal alloys in the dental field. In the present study, we evaluated the load-deflection characteristics of PEEK wires in addition to their frictional properties. Three types of PEEK wires are used: two sizes of rectangular shape, 0.016 × 0.022 in Metal alloys such as stainless steel, cobalt-chromium (Co-Cr), and nickel-titanium (Ni-Ti) alloy are widely used as orthodontic wires. The elastic properties of metal alloys play a significant role in efficient tooth movement in orthodontic treatment . Among tHowever, metal alloy arch wires have the disadvantages of esthetics and metal allergies, so alternatives to metal alloys are needed in orthodontic treatment. Polymer materials have an advantage in esthetics. Some polymers are known to have higher mechanical strengths and are applied in industrial fields. Among them, polyetheretherketone (PEEK) is now attracting attention as an alternative in medical and dental fields. PEEK has a white color and excellent mechanical properties, as well as being known to be bio-inert . TherefoMaekawa et al.  evaluateKasuya et al.  investigIn addition to the load-deflection characteristics, the static friction between orthodontic brackets and wire is an important factor that influences orthodontic tooth movement. Less friction can facilitate tooth movement due to the efficient transmission of orthodontic force to the teeth and the orthodontic treatment period may be shortened without unfavorable anchorage loss or patient pain ,14,15,16In the present study, we evaluated the load-deflection characteristics of PEEK wires of orthodontic size and rectangular and rounded shapes. The frictional properties were also evaluated. Ni-Ti wire was used as a control. The null hypothesis is that PEEK wire is applicable as an orthodontic appliance.p < 0.05). The PEEK wires showed similar load-deflection curves to the Ni-Ti wire except for 16PEEK, which showed a lower bending load as compared to the other two PEEK wires. Slight permanent deformation was found for all three PEEK wires.p < 0.05). A slight amount of permanent deformation was observed for the Ni-Ti wire, and a greater degree of permanent deformation was observed for all three PEEK wires. SL showed different load-deflection curves as compared to EL , as shown in p > 0.05). 16PEEK showed the lowest significant load for any of the three ligation methods (p < 0.05).p < 0.05).The load at the deflection of 1500 μm during unloading was evaluated . This lop < 0.05) and 19-25PEEK showed the highest load among the four wires at the deflection of 1500 μm (p < 0.05). 16PEEK also showed the lowest load deflection of 1500 μm for any of the three ligation methods (p < 0.05).For SL, loads at the deflection of 1500 μm of 16-22PEEK and 19-25PEEK wires were significantly higher than that of Ni-Ti wire (p < 0.05), and the Ni-Ti wire showed the lowest significant permanent deformation (p < 0.05). However, no significant differences were observed in the permanent deformation between the Ni-Ti and three PEEK wires for SL (p > 0.05).Permanent deformation was also influenced by the different ligation methods . Signifip < 0.05).p > 0.05).SEM images of the 16PEEK and the Ni-Ti wires before and after friction tests are shown in In the present study, we evaluated the efficacy of PEEK wires as an alternative to Ni-Ti wire. Three-point bending tests were performed for simulating the early stage of orthodontic treatment, namely, leveling. Ni-Ti wire was used as a control. The load-deflection characteristics and the frictional properties of the PEEK wires were applicable as an orthodontic appliance. Especially, 19-25PEEK wire showed almost totally compatible properties with Ni-Ti wire. Thus, the null hypothesis is accepted.PEEK is classified as super engineering plastic material which has high mechanical strength and chemically inert properties. Thus, PEEK could replace titanium, titanium alloy, Co-Cr-alloys and biological ceramics in orthopedic surgery . For exaAside from esthetic problems, some shortcomings of metallic orthodontic appliances have been reported. For example, conventional metal orthodontic wire and brackets have been demonstrated to release nickel and chromium ions ,21, and Ligation with stainless steel or elastomer is widely applied in orthodontic treatment. Stainless steel requires more skill in the ligation procedure and the ligation conditions are varied according to the clinician. Therefore we did not use stainless steel ligature.Wires were directly held by elastomer in EL. The elasticity of elastomer is expected to give a continuous tightening pressure to the specimen wire in the bracket slot. SL ligation has been introduced for reducing the friction between wires and brackets . For SL,Load-deflection curves indicated that PEEK wires had similar elastic properties to Ni-Ti wire except for 16PEEK in EL. Thicker PEEK wire exhibited a higher load. The use of 16PEEK wire may be avoided for orthodontic use, because it had the lowest load among the tested wires.Comparing the load at a maximum deflection of 2000 μm, 19-25PEEK showed a comparable load with Ni-Ti in SL ligation. The load at the maximum deflection of 2000 μm corresponded to the initial force when placing the wire in the orthodontic bracket at the first stage of orthodontic treatment. The distance of 2000 μm was simulated as the distance of a tooth before moving, according to previous papers ,25. The Stress relaxation tests indicated that the load reduction of 16-22PEEK and 19-25PEEK was larger than that of Ni-Ti. However, 70–80% of the initial load was still maintained and the maintained value was sufficient for orthodontic treatment.The static friction between PEEK wires and brackets was almost the same as that of Ni-Ti wire. For the friction test, EL was employed, in accordance with the previous report ,27. SEM Based on the results of stress relaxation tests after 24 h, it is predicted that there will be no distinct difference in load-deflection behaviors over time between PEEK and Ni-Ti wire. For friction properties over time, frictional properties of PEEK will be maintained because of its high mechanical strength and chemically inert property. Detailed studies for load-deflection and friction properties over times will be needed as a next subject of our study.In the oral conditions, bacterial adhesion and biofilm formation are crucial for dental materials. Hahnel et al.  reportedStaining is another problem in the clinics. Heimer et al.  assessedNonconventional ligature methoOKds such as self-ligation or nonconventional elastomers are now introduced to reduce the friction between wire and the bracket ,32. In t2 (0.406 × 0.559 mm2) and 0.019 × 0.025 in2 (0.48 × 0.635 mm2), and one round shaped wire, diameter 0.016 in2 (0.406 mm2) . As a control, Ni-Ti orthodontic wire with a diameter of 0.016 in.  was used. These wires are referred to as 16-22PEEK, 19-25PEEK, 16PEEK, and Ni-Ti, respectively.PEEK resin was extruded by using specially designed profile dies to create orthodontic wires with clinically relevant cross sections. Three types of PEEK wires were used: two sizes of rectangular shaped wires, 0.016 × 0.022 in2 (0.559 × 0.711 mm2), the mesiodistal width was 2.9 mm, with no built-in torque or tip. The inside of the slot was covered with stainless steel.As brackets, conventional plastic brackets  were used. The slot size was 0.022 × 0.028 inA modified three-point bending system for orthodontics  Figure  was usedIn the present study, the wire was inserted and ligated in the slots of three brackets, which were bonded to each of the three bender rods. This current three-point bending system is an improvement to a previous one . The braFor each ligation method, a comparison was made in the load-deflection curve between the maximum load at a deflection of 2000 μm, the load at a deflection of 1500 μm during unloading, and the maximum permanent deformation after unloading. Each combination of bracket and wire was measured five times. New material was used for each measurement.Modified three-point bending system used in the present study. Bending at a maximum deflection of 2000 μm for three different methods.Each wire was ligated in the brackets by SL, as described above, by using the modified three-point bending system. Then, the wire was deflected at 2000 μm and maintained at the same position at the deflection of 2000 μm for 24 h. The load decrease was monitored during the 24 h. After 24 h, the deflected wire was unloaded and the maximum permanent deformation was evaluated. Measurements for each wire were taken three times.Measurements of the static friction between the orthodontic wire and the bracket were performed according to previous reports in the system shown in The upper end of each 6 cm long wire was connected to the load cell of the universal testing machine and the lower end of the wire was connected to a 150-g weight. The wire was then ligated to the bracket by using EL ligation. Each bracket-wire combination was tested at angulations of 0°. Each wire was drawn through the bracket at a cross-head speed of 20 mm/min for a distance of 5 mm. The static friction between the bracket and the wire was measured as the peak force required to initiate movement of the wire through the bracket. This peak force was defined as the static friction. Measurements were carried out at room temperature and dry conditions. New material was used for each measurement. Each combination of bracket and wire was measured five times. New material was used for each measurement.Before and after the friction tests, the surface conditions of 16PEEK and Ni-Ti were observed by a scanning electron microscope  at an accelerating voltage of 10 kV. Specimens were sputter-coated with Au prior to the SEM observations.p < 0.05, Windows Release 11.5.1, SPSS Inc., Chicago, IL, USA) and Tukey’s tests (p < 0.05). The evaluation of the differences in contact angles was performed by using the non-paired t-test (α = 0.05).For the statistical evaluation, the differences in load and deflection and in the static friction properties were tested by comparing the mean values among the four different wires with one-way ANOVA (Within the limitations of the present study, it is suggested that 19-25PEEK will be applicable for orthodontic treatment with the use of SL ligation as an alternative to Ni-Ti wire. Evaluations of simulated clinical conditions including wet conditions should be further investigated, because eating, brushing or removing the food colorants and stains may influence the load-deflection characteristics and static friction of orthodontic wire in orthodontic treatment.


Aedes aegypti cells and in the mosquito. Employing next-generation sequencing technologies, we identified a total of 126 miRNAs from the Ae. aegypti cell line Aag2. Of these, 13 miRNAs were found to be regulated during CHIKV infection. Putative targets of three of the most significantly regulated miRNAs- miR-100, miR-2b and miR-989 were also analyzed using quantitative PCRs, in cell lines and in mosquitoes, to validate whether they were the targets of the miRNAs. Our study expanded the list of miRNAs known in Ae. aegypti and predicted targets for the significantly regulated miRNAs. Further analysis of some of these targets revealed that ubiquitin-related modifier is a target of miRNA miR-2b and plays a significant role in chikungunya replication.Arboviruses that replicate in mosquitoes activate innate immune response within mosquitoes. Regulatory non-coding microRNAs (miRNA) are known to be modulated in mosquitoes during chikungunya infection. However, information about targets of these miRNAs is scant. The present study was aimed to identify and analyze targets of miRNAs that are regulated during chikungunya virus (CHIKV) replication in  The response is executed via several mechanisms such as phagocytosis2, antimicrobial peptides3, melanotic encapsulation4, and nitrogen intermediates5. In addition to these mechanisms, insects employ other pathways such as RNA interference (RNAi), immune deficiency (IMD), and toll and JAK/STAT signaling pathways to provide defense against pathogens, including viruses7. Amongst viruses, some are transmitted by arthropods and are called arboviruses, many of which are of importance due to the impact they have on human health8. Mostly transmitted by the Ae. aegypti and Culex tritaeniorhynchus mosquitoes, these viruses are characterized by their requirement to alternate between a vertebrate host and the mosquito.Innate immunity in insects has been described as the germ line-encoded anti-infection response of the hostAlphavirus, family Togaviridae. Its genome is a single-stranded positive-sense RNA that transcribes mRNA from two open reading frames (ORFs): one that expresses nonstructural proteins and another that expresses structural proteins. In addition to these two ORFs, the genome contains a 5′-terminal cap and a 3′-terminal poly(A) tract along with 5′ and 3′ untranslated regions (UTRs) that contain signals important for replication of the RNA9. Whereas much information is available with respect to the innate and adaptive immune response of CHIKV in the mammalian hosts11, little is known about the innate immune responses in the vector during CHIKV replication12. Amongst all innate immune responses, RNAi assumes the first level of defense against RNA viruses in mosquitoes.Chikungunya virus (CHIKV) is an arbovirus belonging to the genus 13. Whereas exogenous RNAi is triggered by the presence of siRNA generated through the processing of dsRNA and virus-derived siRNAs (vsiRNAs), another class of small RNAs that are regulatory are the microRNAs (miRNA)14. A class of small, non-coding RNAs of 19–24 nt in length, miRNAs regulate gene expression post-transcriptionally by binding to complementary regions in the 3′ UTR of target mRNAs15. Reports have revealed the role of miRNAs in several cellular processes16, including regulating virus replication17. However, studies on the cellular targets through which these miRNAs may act are scant, thereby not providing enough information on the mode of action of miRNAs in insect immunity.RNAi is a conserved, sequence-specific, gene-silencing phenomenon that is induced by double-stranded RNA. In the case of a viral infection in insects, viral replication intermediates in the form of double-stranded RNA (dsRNA) trigger RNAi that acts as a defense mechanismAedes albopictus cells regulated vector miRNAs18. The present study was initiated to study the impact of the cellular targets of some of the miRNAs that are regulated in Ae. aegypti. Upon CHIKV infection in an Ae. aegypti cell line, namely Aag2, significantly regulated miRNAs were identified through next-generation sequencing, targets of the significantly regulated miRNAs were predicted through computational approaches, and pathway analysis of these targets were performed. Furthermore, through loss-of-function assays, targets of the selected miRNAs were validated using quantitative PCR, both in Ae. aegypti cells and in Ae. aegypti mosquitoes. The present study also showed that miR-2b targets the 3′UTR of ubiquitin-related modifier (URM) to control CHIKV replication.A recent study by our group revealed that CHIKV replication in Ae. aegypti was performed using high-throughput small RNA sequencing. Small RNA libraries obtained from a CHIKV-infected Ae. aegypti cell line (Aag2) at 12 and 24 hours post-infection (h.p.i.) were compared with Ae. aegypti miRNAs from an uninfected Aag2 cell line. These time points were chosen in order to evaluate the vector miRNAs during early-phase CHIKV infection. Post-sequencing, small RNA reads from all the three libraries were separately mapped on the Ae. aegypti genome. The mapping showed 76.12% reads of uninfected library, 82.34% reads of 12 h.p.i. library, and 51.63% reads of 24 h.p.i. library. The mapped reads were further used for the identification of known miRNAs, including up to one mismatch using data analysis pipeline established in-house18. Unmapped reads of 12 and 24 h.p.i. libraries were further mapped on to the CHIKV genome. Details of small RNA analysis of the datasets are shown in Supplementary Tables Global miRNA profiling of . Log-fold change and p-value of the predicted miRNAs were calculated using edgeR packages  described elsewhere18. miRNAs with p-value ≤ 0.05 were considered significant. The analysis revealed a total of thirteen miRNAs to be significant in all analyses . Three miRNAs  were found to be significantly regulated (p ≤ 0.05) in the 12 h.p.i. library when compared with the uninfected library: two miRNAs (miR-10-5p and miR-2b) were upregulated, and one miRNA (miR-2951-5p) was found to be downregulated. Similarly, eleven miRNAs were significantly regulated in the 24 h.p.i. library, with five miRNAs  showing overexpression and six miRNAs  being underexpressed.To analyze the relative abundance and expression profiling of miRNAs, tags per million (TPM) of total RNA reads for each miRNA in all libraries were calculated. The TPM values were compared between libraries and a heat map of all predicted miRNAs was generated and visualized. We normalized the row values to have mean of 0 and a variance of 1 Fig. . Log-foses Fig. . Three p < 0.001), miR-184-3p showed a 7.5-fold increase (p < 0.0001), miR-71-5p showed a 7.7-fold increase (p < 0.0001), whereas changes in the expression profiles of miR-278-5p, miR-989, and miR-100 remained statistically insignificant  analysis for randomly picked six miRNAs. Four out of the six miRNAs showed expression profiles similar to those observed in small RNA sequencing analysis, whereas miR-100 and miR-71-5p showed different patterns. Quantitative RT-PCR showed that at 24 h.p.i., miR-989 and miR-71-5p were downregulated and miR-2b, miR-184-3p, and miR-278-5p along with miR-2b were upregulated. This scenario changed at 48 h.p.i. in the case of miR-2b, miR-184-3p, and miR-71-5p: these miRNAs showed significant upregulation in their expression at 48 h.p.i. miR-2b showed a six-fold increase .To understand the function of miRNAs, it is important to study their targets. miRNAs are known to function in clusters to regulate various biological processesIn order to confirm the targets that were predicted, we selected three miRNAs, namely, miR-2b, miR-100, and miR-989 for target prediction, that were among the most significantly differentially expressed in small RNA sequencing, further downstream validations and biological relevance in other systems. A total of 785, 1024, and 294 targets were predicted for miR-2b, miR-989, and miR-100, respectively. Further analysis was performed on the basis of the involvement of targets in immune and signaling pathways, and their binding energies and two targets for each of the three miRNAs were validated further. For miR-2b, two genes, namely, URM (AAEL008680) and ubiquitin (AAEL006511), were selected on the basis of statistical significance and their relevance in immune-related and signaling pathways. In the case of miR-100, cdc42 (AAEL011500) and sumo-ligase (AAEL015099) were selected. Similarly, for miR-989, sh2/sh3 adaptor (AAEL013539) and vacuolar ATP synthase (AAEL012819) were selected for validation.Ae. aegypti cells or by injecting in the mosquitoes, and the time points that exhibit maximum silencing were ascertained . On the basis of these findings, we hypothesized that URM is a target of miR-2b. To confirm this, we performed a luciferase assay in HEK 293-T cells to evaluate the binding of miR-2b to the 3′UTR of URM. Empty pmR-mCherry vector and pmirGLO vector containing 3′UTR of URM served as controls; sequence-specific binding of miR-2b to the 3′UTR of URM was evaluated by mutating the miRNA-binding site at the 3′UTR and then the assay was performed. The assay revealed a >80% reduction in the luciferase activity in those constructs with wild-type 3′UTR of URM. However, constructs with a mutated binding site showed loss of binding, and the luciferase activity reverted to control values. Taken together, our results establish that URM is indeed a target of miR-2b . We observed an increase in viral replication when miR-2b is silenced, thereby proving that miR-2b may be playing a role in regulating CHIKV infection in Ae. aegypti. Next, we sought to understand the role of URM during CHIKV infection, as it is a target of miR-2b. We first checked the expression of URM in Ae. aegypti mosquitoes 24 and 48 h after CHIKV infection. We observed more than a 2.5-fold reduction in URM expression upon CHIKV infection, which further reduced in a day-dependent manner  in CHIKV viral genomic RNA in 24 h and a more drastic 87% (p < 0.0001) at 48 h.p.i., whereas in GFP dsRNA-transfected cells that served as the control, the CHIKV viral genomic RNA remained similar to control infection . This finding was surprising, as we hypothesized that a reduction in URM would increase CHIKV viral genomic RNA, as the presence of virus seems to reduce the expression of URM, as can be observed in Fig. p = 0.005) when the miRNA-silenced mosquitoes were infected with CHIKV .To understand the role of URM during CHIKV infection better, we dissected the expression pattern of this transcript during CHIKV infection in the presence and absence of miR-2b. As a first step, we confirmed whether silencing miR-2b regulated CHIKV infection. For this purpose, we inhibited miR-2b using antagomir in the Aag2 cell line and infected it with CHIKV virus. After 24 hours of infection, we checked the CHIKV viral genomic RNA and found that inhibiting miR-2b increased CHIKV replication Fig. . We obsner Fig. . This fiion Fig. . This fIKV Fig. .Figure Ae. aegypti spreads many viral diseases such as dengue and chikungunya and has more recently created global havoc with Zika transmission, causing much harm to healthcare and public health management. The most effective way of controlling these infections is through vector control. Central to vector control is the understanding of mechanisms that the vector employs to tackle the viruses it carries. Viral infection creates a competition between the vector and the virus in taking over the cellular machinery to run each of their own regime and successfully establish each of their hold. During this process, the host activates genes of several pathways to control the virus and the virus attempts to either activate the factors that help in membrane budding, which in turn will help its replication, or suppress the pathways inhibiting its replication and expansion25. The purpose of our present study was to identify the cellular factors that may play a role in viral replication. One approach is to identify the vector factors that may be regulated upon infection using several methodologies identifying host factors that regulate viral infection26. Another approach is to identify those regulators that regulate these factors within the cell, such as miRNAs, and then identify their targets22. These targets may further be validated for their functions to understand their mode of action during specific conditions. The second approach provides information at a more biological level, offering insights both into cellular transcripts and into their modulators such as miRNAs that could be regulating them in a more centralistic manner. Therefore, we utilized the second method in the present study.Ae. aegypti had identified 86 distinct miRNAs27; our data expanded the list of known miRNAs to 124. Whereas the earlier study used Roche 454 platform for generating high-throughput data, our study utilized Ilumina sequencing, generating much more data, which could be a plausible reason for the identification of more number of miRNAs. The new miRNAs have already been described in earlier studies on Ae. albopictus28, thereby proving that these are bonafide miRNAs.An earlier report on Ae. albopictus and Ae. aegypti has been previously studied30. miRNAs regulated in these studies were found to be regulated in the present study as well. For instance, miR-285 was reported to be among the highest expressing miRNAs in the saliva of CHIKV-infected Ae. aegypti30. Similarly, miR-317 and miR-2951-3p were found to be upregulated during dengue virus infection in Ae. albopictus31 and miR-34-3p was shown to be involved in Wolbachia infection32. Whereas our earlier study on Ae. albopictus miRNAs highlighted miR-2b and miR-100 to be regulated upon CHIKV infection18, miR-100 has been shown to attenuate human cytomegalovirus replication via the mTOR pathway33 and have also been implicated in pathways related to cell cycle progression with targets such as plk1, while also playing a role in apoptosis34. Similarly, miR-989 has been predicted to be an important miRNA targeting insect immunity in other mosquito species as well36. The time points of our study coincided with the early replication events of CHIKV in the vector. Studies have shown that replication kinetics of the virus differ in the host and the vector, with the virus replicating at a much slower pace in the vector than in the host, probably allowing for persistent infection in the vector to enable efficient virus transmission throughout the life of the vector9. In addition to the several mechanisms that may be employed by the virus, host-derived factors may also play a role. Due to the significance of miRNAs in mosquito biology, we hypothesized that vector miRNAs and their targets could also be involved in regulating viral replication.Regulation of miRNAs during arboviral replication in 37. Prediction tools identified a total of 8928 targets for all the regulated miRNAs taken together; on the basis of binding energies and relevant pathways, a total of six targets were chosen for the three miRNAs, to study whether the expression of these transcripts was affected by silencing the respective miRNAs that supposedly target them. Loss-of-function and gain-of-function analyses have been earlier employed to study the role of miRNAs in mosquitoes38. In the present study, out of the six transcripts, only one target, URM, showed promising results, clearly emphasizing the importance of validating miRNA targets by wet lab validations to confirm that the candidate is indeed a target of miRNA. Whereas luciferase assays provide information on binding of the miRNA to the 3′UTR of the target gene, the expression profile of the putative targets provides indirect information of the action of these miRNAs on the targets39.Using RNAhybrid, multiple targets for the selected miRNA were predicted, taking the seed binding region into consideration. The targets were analyzed by Gene Ontology (GO) terms and selected for further validation. Bioinformatics has been useful in identifying targets of miRNAs in the recent pastin vitro conditions and mosquitoes. In the case of miR-100, of the two targets chosen (sumoligase and cdc42), cdc42 was found to be regulated in the cell line; however, we did not observe any significant regulation in the mosquito. Sumoligase expression was, however, almost negligibly regulated by the knockdown of miR-100. Cdc42 is a highly conserved small GTPase of the Rho family, acting as molecular switch in a wide range of signaling pathways such as vesicle trafficking and cell polarity40; sumoligase has been shown to play a role in immunity to virus infection41. Neuraminidase to the host cell surface is also regulated by cdc42 affecting influenza virus replication42. The miR-989-predicted target sh2/sh343 and vacuolar ATP synthase44 have been found to play a role in viral infections. But sh2/sh3 adaptor and vacuolar ATP synthase were not found to be regulated when the cells or mosquitoes were transfected or injected with the respective antagomir, negating the possibility of them being a target of miR-989. Targets of miR-2b showed the most promising results. URM and ubiquitin were selected, as both of these targets have been previously reported to be significant in viral replication and infection46. URMs such as ISG15 and FAT-10 have been the shown to play a role in innate immunity and viral infection48. In our study, we found URM to be differentially regulated upon its antagomir treatment in Ae. aegypti cell line and Ae. aegypti mosquito. The expression level of URM increased 24 h after transfection with antagomir both in the cell line and in the mosquitoes. In the case of mosquitoes, there was more than 20-fold increase in its expression after 48 h of miRNA silencing, clearly emphasizing the effect miR-2b has on the target. To further validate the target of miR-2b, we performed a luciferase reporter assay that shows significant binding to the 3′UTR of URM. Most significant in our findings was the pattern of URM expression during CHIKV infection and the manner in which it was altered when miR-2b was silenced in mosquitoes.In our study, we tested the effect of silencing of miRNAs on their putative targets in both Ae. aegypti by miR-2b through its regulation of a host factor, namely, URM  was maintained at 28 °C and 5% CO2 in Schneider media  supplemented with 10% FBS and antibiotics. The cells were infected with MOI 10 of a CHIKV isolate (accession no. JF950631.1) that was characterized during an outbreak in 201052. The cells were infected in triplicates and harvested 12 and 24 h.p.i and processed further for small RNA sequencing.Ae. aegypti eggs were allowed to hatch into first instar larvae which were fed on fish food until fourth instar. Upon development, the pupae were transferred to water-filled plastic containers inside cloth cages and allowed to emerge. Upon emergence, the adult mosquitoes were reared under controlled conditions at 28 ± 2 °C, 70–75% humidity, and fed with 2% sterile glucose solution. Ae. aegypti mosquitoes were infected by blood-feeding using 500 μL of polyethylene glycol purified CHIKV mixed in 2 mL of rabbit blood. Previously starved mosquitoes were fed for 20 min at room temperature through membrane feeding. After blood feeding, fully fed mosquitoes were separated and taken for further processing for time point studies. All experiments related to mosquitoes and virus were performed in bio-containment facilities ACL-2 and BSL-3, respectively.CHIKV-infected Aag2 cells were harvested at 12 and 24 h.p.i. and stored in Trizol  until RNA extraction. Small RNA population from the total RNA was enriched and extracted using miRNeasy kit  as per manufacturer protocol. Agilent 2100 Bioanalyzer RNA Nano 6000 kit was used to check the quantity and quality of isolated RNA. Uninfected and infected samples were outsourced for small RNA sequencing by Illumina Genome Analyzer II .18. The pipeline makes use of tools like Bowtie, RNAfold, and RNAplot for analysing the data55. Briefly, Ae. aegypti genome and coding region sequences were downloaded from VectorBase using the BioMart tool56. Pre-miRNA and mature miRNA sequences of arthropods were downloaded from the miRBase database V.1957. Other non-coding RNAs (ncRNA) sequences were fetched from the ncRNA database58. The downloaded sequences were indexed separately. After a quality check and adaptor trimming of small RNA reads, reads with length ≥18 nt were matched to mature miRNA sequences to identify known miRNAs. Tags per million (TPM) values were calculated for matched miRNAs. UTR sequences of Ae. aegypti genes were downloaded from VectorBase and targets of significant miRNAs were predicted using RNAhybrid tool59. The targets were filtered on the basis of complementarities of the miRNAs with the targets and energy of the miRNA:target, i.e, not exceeding −20 kcal/mol. Functional enrichment of the predicted targets was performed by WEGO webtool60 using GO term accession of each target gene fetched from VectorBase using the BioMart tool. The dataset analysed during the current study are available in ArrayExpress (accession number E-MTAB-5222).Data analysis was carried out using an in-house PERL-based pipeline developed for analysing small RNA data as described elsewhere6 Aag2 cells were seeded into 6-well plates to reach 75% confluency before proceeding to transfection. A volume of 100 pmol of antagomir for each miRNA, miR-2b, miR-100, and miR-989, along with the negative control was transfected and the cells were incubated at 28 °C for 4 h before changing with fresh media. The cells were collected 24, 48, and 72 h after transfection and processed for RNA isolation. Aag2 cells that were transfected with miR-2b antagomir were infected with CHIKV after 24 h and cells were collected 24 h.p.i for RNA isolation.Antagomirs for miR-989, miR-100, and miR-2b complementary to mature miRNA sequence were synthesized from Ambion . In addition, scrambled miRNA provided by the manufacturer was used as the negative control in all experiments. Antagomir transfections were performed with Attractene transfection reagent  in Aag2 cell line as per the manufacturer instructions. Briefly, 0.3 × 10Female mosquitoes (4–5 days old) were divided into four batches of 100 mosquitoes each. The first batch was injected with 69 nL of negative control (scrambled miRNA). The second, third, and fourth batches of mosquitoes were injected, with 69 nL of 100 µM miR-2b, miR-100, and miR-989 antagomirs, respectively. Mosquitoes were collected at two time points, 24 and 48 h, and were stored in Trizol  at −80 °C until RNA extraction. Time-wise knockdown of miRNA expression following injection was checked by miRNA qRT-PCR. In the case of subsequent blood feeding studies, nanoinjected mosquitoes were allowed to revive for 24 h prior to membrane feeding. Every experiment was repeated at least three times.Ae. aegypti genome using Bowtie, and 250 bp flanking region from each side were extracted using in-house PERL script. miRNA miR-2b binding site 3′UTR of URM were amplified with primers containing the desired mutation for binding sites of miRNA miR-2b and cloned into the pmirGLO vector  for lucifersae assay. Pre-miRNAs were PCR amplified and cloned into pmR-mCherry vector  along with the 3′UTR of CHIKV which was cloned into pmirGLO vector .All the mature miRNAs were mapped against Luciferase activities were measured with a luminometer according to manufacturer recommendation  24 h after transfection using the Dual-Glo luciferase reporter assay system . Renilla luciferase activity was normalized using firefly luciferase activity for each sample.in vitro transcribed with T7 and SP6 polymerase using Promega’s Riboprobe combination kit . Further dsRNAs were purified using the Trizol  method described earlier and stored until further use. For dsRNA-mediated silencing, 800 ng of total dsRNA was injected into a mosquito. In the case of subsequent blood-feeding studies, nanoinjected mosquitoes were allowed to recover for 24 h prior to membrane feeding. For time-series experiments, female mosquitoes were collected 24 and 48 h after feeding and stored in Trizol  at −80 °C until RNA extraction.For dsRNA preparation, URM and green fluorescent protein  were cloned into pGEMT-easy vector  and were Antagomir-injected mosquitoes were collected 24 and 48 h after injection and Aag2 cell line 24, 48, and 72 h after transfection. RNA was isolated by the Trizol  method and kept at −80 °C until further use. The isolated RNA was used for qPCR reaction for the miRNA and host factor-expression profiling.−ΔΔCT method. Details of the qRT-PCR primers used in the study are provided in Supplementary Table Expression profiling was carried out by quantitative RT-PCR. For miRNAs, 1 µg of RNA was polyadenylated, reverse transcribed, and quantified by qRT-PCR using NCode miRNA First Strand cDNA Synthesis and qRT-PCR kit . qRT-PCR reactions were set up using 1:10 diluted cDNA as template following manufacturer instructions. For other transcripts, cDNA was used directly for quantitative RT-PCR using QuantiTect SYBR Green RT-PCR Kit . Experiments were conducted a minimum of two times, with each experiment set up in triplicates. For miRNA and transcript expression profiling, 5.8 s rRNA was used as an endogenous control. Expression levels were then calculated using the 2p-value cut-off was determined from the data, with the significance threshold selected as 0.05. Statistical analysis of experimental data was conducted using GraphPad Prism (version 5) using Student’s t-test when comparing two conditions. Data from different treatments were subjected to analysis of variance (ANOVA). For multiple comparisons, Dunnett’s test was performed. Values of p < 0.05 considered significant have been represented with an asterisk in the figures.Differentially expressed significant miRNAs were predicted using edgeR package in R. The Small RNA sequencing data were deposited in ArrayExpress (accession no. E-MTAB-5222).Supplementary Information


When a patient has cone beam computed tomography (CBCT) images based on the treatment plan, it is possible to use these images for evaluation of caries, and there is no need for new radiographs, according to the “as low as reasonably achievable” (ALARA) principle. The aim of this study was to determine the effect of filtration and thickness of CBCT cross-sections on detection of proximal caries.In this in-vitro study, 100 teeth were placed in the dental sockets of a dry skull, and were fixed in normal proximal contacts. CBCT images were taken and were evaluated by two observers on the panoramic view at 1-, 3- and 5-mm-thick cross-sections, with the use of filtrations 0, 1 and 2. Afterwards, the samples were sectioned and underwent a histological evaluation. McNemar’s test was used to compare the findings on CBCT images and histological evaluation. Receiver operating characteristic (ROC) curves and logistic regression were used to evaluate the diagnostic accuracy of different cross-sections.The maximum AZ-value was achieved at 3-mm thickness/filtration 2. However, the differences between 1-mm thickness/filtration 2 and 1-mm thickness/filtration 1 were not significant . The minimum AZ-value was achieved at 5-mm thickness/filtration 0.Although CBCT is not sufficiently effective in detecting caries, the best cross-sections for detection of proximal caries were achieved at 3-mm thickness/filtration 2, 1-mm thickness/filtration 2 and 1-mm thickness/filtration 1. Dental caries is the most prevalent clinical problem in dentistry and has a high incidence rate in the population . TherefoHowever, diagnosis of interproximal caries in posterior teeth is still a challenge in dentistry, and it usually requires a combination of clinical and radiographic examinations ,7. In reThis new technique helps dental practitioners to collect data and improve treatment planning with the use of three-dimensional (3D) images at axial, sagittal and coronal cross-sections . CBCT teIn the present in-vitro study, 100 human teeth, consisting of 20 canines, 40 premolars and 40 molars with or without proximal caries were selected from among 400 teeth, using simple random technique. The teeth had been extracted for periodontal or orthodontic reasons at the School of Dentistry, Isfahan University of Medical Sciences. The teeth were cleaned of all the calculi and debris using an ultrasonic device , were disinfected in 2% sodium hypochlorite (NaOCl) solution for 20 minutes, and were stored in distilled water. Then, the crowns were separated from the roots using a #835 bur . The dental crowns were divided into 20 groups (n=5). Ten groups included maxillary teeth, and the rest included mandibular teeth. Each group consisted of a canine, first and second premolars and first and second molars in one quadrant. Five dry skulls were prepared, and 4 groups of teeth were placed in the alveolar sockets of each skull so that the teeth in each group were placed in their real anatomical positions, with proper proximal contacts in the maxillary and mandibular alveolar sockets. Finally, the teeth were fixed in their sockets with red utility wax. The placement was done in a way that each row was in a good proximal contact. The mandibular and maxillary teeth were occluded together, and were fixed by wax , . To simuIn the next stage, CBCT images were taken with the use of Soredex CBCT unit . Each skull was fixed in the CBCT system with the use of ear rods and was scanned for 12.6 seconds at kilovoltage peak (kVp)=89 and milliampere (mA)=6. The images were reconstructed with the system’s software program . Definitively cariousPossibly cariousNot definitive and suspectedPossibly not cariousDefinitively not cariousThe images were evaluated by two oral and maxillofacial radiologists with 6 years of experience in a random single-blind manner. The images were visualized on a 22-inch 32-bit LG monitor  with a resolution of 1440×690 pixels, using the CBCT system’s software program in a dimly lit room. The observers scored the images on the panoramic view  with three different filtrations (resolutions) of 0, 1 and 2, and three different cross-section thicknesses of 1, 3 and 5 mm, based on the presence or absence of proximal cares in a 5-point scale , as follIn order to evaluate intra-observer agreement, the observers evaluated and scored the images twice with an interval of two weeks, in order to eliminate the effect of memory. Subsequently, the teeth were sectioned mesiodistally, parallel to the tooth long axis with a mean cross-section thickness of 0.4mm, using Accutom cutting instrument .No caries in the proximal surfaceProximal caries in enamelProximal caries extending to the dentino-enamel junction (DEJ) or located in the external half of dentinEach cross-section was evaluated by a pathologist under a stereomicroscope at ×15 magnification. Each white demineralized lesion or yellow-brown discoloration in enamel or dentin was considered a carious lesion in the histopathological view [0.10 <: No agreement0.11–0.4: Poor agreement0.41–0.60: Acceptable agreement0.61–0.80: Strong agreement0.81–1.0: Excellent agreementKappa agreement coefficient was calculated for intra- and inter-observer agreement, and was classified as follows:In order to evaluate the accuracy of caries detection in different radiologic cross-sections, the results reported by the radiologists were compared with the histopathology findings using complete and absolute sensitivity, specificity and the receiver operating characteristics (ROC) analysis. Values under the ROC curve  were calculated for different cross-sections using SPSS 22 software program . Wilson confidence interval-based comparison was used to evaluate the differences in the accuracy of different cross-sections (α = 0.05).1-mm thickness/filtration0 (P<0.001), 3-mm thickness/filtration0 (P<0.001), 3-mm thickness/filtration1 (P<0.001), 5-mm thickness/filtration1 (P<0.001) and 5-mm thickness/filtration2 (P=0.046).The results indicated a direct correlation between filtration and correct diagnosis in CBCT, and a reverse correlation between the thickness of view and correct diagnosis in CBCT. However, there were no significant differences between 3-mm thickness/filtration 2 and 1-mm thickness/filtration 1 (P=0.617), or between 3-mm thickness/filtration 2 and 1-mm thickness/filtration 2 (P=0.459). The most inaccurate diagnoses in all the cross-sections were related to the teeth with enamel caries except for 1-mm thickness/filtration 0, in which caries-free teeth were more accurately diagnosed compared to the teeth with carious lesions in the external half of dentin.CBCT is a new imaging technique in dentistry. All the new diagnostic methods should be compared with the imaging techniques that are currently used in the clinic. Although there are some studies available in this regard, there are still some debates in relation to the efficacy of the CBCT technique .Akdeniz et al  reportedAn in-vitro study by Haiter-Neto et al  showed aCharuakkra et al  reportedTherefore, the increase of thickness decreases the number of correct diagnoses, whereas the increase of filtration, increases the number of true diagnoses in CBCT. In this study, ROC curves were used to evaluate the diagnostic accuracy of different cross-section thicknesses and filtration rates. ROC analysis is a more comprehensive parameter for evaluation of diagnostic accuracy compared to sensitivity and specificity .In addition, ROC analysis is a more eloquent parameter for comparison of the diagnostic performance of two or more diagnostic techniques, because it is less significantly affected by the inherent ability of the observer to interpret images, and has been used in various studies ,24. In tIn addition, a limited number of cross-sections was evaluated in the present study to detect caries. Another limitation of the present study was the fact that this study has been carried out in vitro under ideal conditions, and non-moving objects were radiographed in the absence of metallic restorations and tissues around the teeth, which might complicate detection of caries in vivo.Within the limitations of the present study, it has been concluded that CBCT technique is not accurate enough for evaluation of proximal caries. The best cross-sections for evaluation of caries were achieved at 3-mm thickness/filtration 2, 1-mm thickness/filtration 1 and 1-mm thickness/filtration 2.


Previously, we have identified biomarker genes expressed in the lungs for evaluation of influenza vaccine safety, and their usefulness in rodent models and for adjuvant-containing vaccines has already been reported. Here, our purpose was to develop a novel humanized mouse model retaining human innate-immunity–related cells to assess the safety of influenza vaccines using the previously identified biomarker genes. In the present study, we tested whether the two humanized models, a short-term and long-term reconstitution model of NOD/Shi-scid nullIL2rγ mice, are suitable for biomarker gene–based safety evaluation. In the short-term model, human CD14+ cells, plasmacytoid dendritic cells, CD4+ and CD8+ T cells, and B cells were retained in the lungs. Among these cells, human CD14+ cells and plasmacytoid dendritic cells were not detected in the lungs of the long-term model. After the vaccination, the expression levels of human biomarker genes were elevated only in the short-term model when the toxicity reference vaccine was inoculated. This phenomenon was not observed in the long-term model. The levels of human cytokines and chemokines in the lungs increased in response to the toxicity reference vaccine in the short-term mouse model. According to these results, the short-term model provides a better platform for evaluating vaccine safety in terms of human peripheral blood mononuclear cell–mediated initial reactions in vivo.Safety evaluation of a human vaccine is critical for vaccine development and for preventing an unexpected adverse reaction in humans. Nonetheless, to date, very few systems have been described for preclinical studies of human adverse reactions  Vaccination is an effective method for prevention of infectious diseases and has been widely used by many people around the world. Vaccines are inoculated into healthy people, including children and elderly subjects. Therefore, a safety evaluation system is needed to predict or evaluate adverse reactions caused by vaccines in humans.Adverse reactions caused by vaccination, especially pain at the site of inoculation, swelling, and fever, are thought to be due to the initial biological response caused by an endotoxin, vaccine antigen, or adjuvants . These rin vivo. Humanized mouse models have been expected to serve as a useful preclinical tool for assessment of immune responses in vivo [SCID/IL2rγKO (NOG and NSG) mice has been reported involving either human CD34+ stem cells or peripheral blood mononuclear cells (PBMCs) [+ cells, and therefore, it takes a long time to prepare such a model. Our aim was to create a tool for evaluation of vaccine safety  as a simple method. Martino et al. reported that intravenously injected human PBMCs are transported into lungs within a few hours [et al. reported that immunodeficient NOJ (NOD/Scid/nullJak3) mice that were injected with human PBMCs at 24 h prior to the vaccination showed elevation of vaccination-dependent production of antigen-specific IgG, suggesting that APCs are recruited within 24–48 h after PBMC engraftment [To date, very few systems have been described for preclinical research that reproduce human reactions  in vivo , but cha (PBMCs) –9. In th (PBMCs) –11. Neve (PBMCs) –13. Thesew hours , and Wadraftment . As statnullIL-2Rγ mutation into conventional SCID and nullRag1/2 mice. These strains showed extremely high engraftment rates and differentiation of human cells, resulting in remarkable advances in the development of human disease models. Data accumulated to date suggest that NOG/NSG mice are the best recipients of a humanized tissue, and the success of human-cell engraftment can be ranked in the following order: NSG = NOG > NOD.Cg-tm1Mom Il2rgtm1Wjl /SzJRag  (NRG) > BALB/c-null Il2rgnullRag2 (BRG) > NOD.CB17scid-Prkdc/J (NOD/SCID) > C57BL/6-null Il2rγnullRag2 (B6RG) [in vivo. We show that the marker genes of the influenza vaccine safety can predict adverse reactions in humans.In this study, we attempted to evaluate influenza vaccine safety in humanized mouse models to demonstrate applicability of the above-mentioned marker genes to humans. In the 2000s, a series of immunodeficient mice was developed by introducing the l (B6RG) , 17. The+ T cells, 7.8% for CD8+ T cells, 5.0% for CD19+ B cells, 17.7% for monocytes (CD14+ cells), and 0.11% for pDCs among all PBMCs , monocytes, and pDCs by fluorescence-activated cell sorting (FACS). The frequencies of cell types were 52% for CD4s Figure  in agree7 cells/mouse as described in another study [7 cells/mouse, and the model was named the “long-term model (LT-model).” The schemes of the experiments are depicted in Figure To develop humanized mouse models, human PBMCs were intravenously injected into NOG mice at 2.0 × 10er study ; this moTo assess the lesions in organs, we analyzed blood biochemical and histopathological changes of the following organs: the spleen, liver, and lungs. Furthermore, to verify the differences in toxic reactions between the humanized mice and wild-type mice, BALB/c mice were also subjected to blood biochemical analyses and lung histopathological examination. Serum blood urea nitrogen (BUN) and creatinine (CRE) served as indicators of kidney functions; creatinine kinase (CPK) served as an indicator of muscle damage; alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (T-Bil) were used as indicators of liver damage; lactate dehydrogenase (LDH) served as an indicator of tissue damage; C-reactive protein (CRP) was an indicator of inflammatory reactions; and surfactant protein-D (SP-D) served as an indicator of lung damage. The blood biochemical analyses showed that no significant changes were induced by vaccination in either the ST- or LT-model Figure . In addi+ or CD8+ T cells were detected in PB; however, CD19+ cells were scarce . These results indicated that both the ST- and LT-model retained human T cells systemically.To test whether human PBMCs were retained and repopulated peripheral blood (PB) and spleen in the humanized mice, flow-cytometric analyses of the lungs at 16 h after vaccination were performed on both models. In the ST-model, CD4e Figure . Of notee Figure . Likewisy Figure . In the e Figure . The nume Figure . In the d Figure . In addil Figure . In PB a−, CD14−/mid, HLA-DR+, CD123+, and CD11clow cells indicating pDCs were detected , and CD14+/ SSChigh cells were also detected; however, CD19+ cells were scarce. The retained levels of these lymphocytes were not changed by vaccination  have been limited in terms of applicability to humans judging by the obtained results. In the presented models, both human PBMC-mediated reactions and informative data on the expression levels of marker genes are seen. Thus, the proposed human-PBL model may offer the benefit of safety evaluation of influenza vaccines in humans. In addition, the possible benefit of using the human-PBL model is that it can provide information about both human and mouse marker genes. In particular, the lungs in the human-PBL model contain human PBMCs, mouse PBMCs, and mouse parenchymal cells . As shown in In the human-PBL model, as compared to one study on rats , signifi+/SSChigh cells in the lungs, and this humanized mouse model may be useful for the human safety evaluation of influenza vaccines by means of human marker genes. In addition, our present data indicate that some of the marker genes can be applicable to humans, and we expect that this information can form the basis for the development of novel methods for assessment and prediction of human safety of influenza vaccines. Because commercially available PBMCs were used in this study, we could not show whether a vaccination history or infection history can be useful for this evaluation system. In the future, if it becomes possible to evaluate the history of a PBMC donor , such data will make this model more robust.In conclusion, in this study, we for the first time developed a short-term model involving pDCs and CD 14scid-IL2rgnull  mice were purchased from In-Vivo Science, Inc. , and female 6- to 7-week-old BALB/c mice (16–20 g) were obtained from SLC , and maintained under specific pathogen-free conditions. All the mice were housed in rooms maintained at 23 ± 1°C and 50% ± 10% relative humidity, in a 12 h light/dark cycle, and were provided with sterilized food and water ad libitum. The mice were subjected to experiments at the age of 7 weeks and were acclimated for at least 7 days before the experiments. All the animal experiments were performed according to the Declaration of Helsinki and the guidelines of the Institutional Animal Care and Use Committee of the National Institute of Infectious Diseases (NIID), Tokyo, Japan. The study protocol was approved by the Institutional Animal Care and Use Committee of NIID.Female 6- to 7-week-old NOD/Shi-Human PBMCs from one healthy donor  were obtained from AllCells  and were subjected to the experiment shown in Figures 7 cells/ml and transplanted via the tail vein into NOG mice at 4 × 107 cells/mouse to create the ST-model. To set up the LT-model, 107 PBMCs were intraperitoneally injected into NOG mice. The mice were subjected to the vaccination experiment at 3 weeks after the PBMC engraftment. This is because more than 3 weeks later, the mice will develop graft-versus-host disease according to our preliminary study (data not shown). The experiments on human blood samples were approved by the Institutional Ethics Committee on Human Experimentation and were conducted in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan.Prior to the engraftment, the PBMCs were resuspended in the RPMI 1640 medium supplemented with 10% of fetal calf serum (FCS) at a density of 4 × 10The red blood cells were lysed with an ammonium chloride solution, and mononuclear cells were prepared as single-cell suspensions in 5% FCS-supplemented PBS. In particular, lungs and spleen were treated with 1 mg/ml type IV collagenase  in Hanks' balanced salt solution at 37°C for 30 min. The digestion reactions were stopped by adding EDTA (10 mM) and placement on ice. Mononuclear cells were incubated for 30 min at 4°C in the dark with the appropriate antibodies . After wRE is a national toxicity reference issued by NIID (Japan). RE is a lyophilized whole-virion preparation of an inactivated influenza virus and consists of the three strains of inactivated whole-virions: A/Newcaledonia/20/99 (H1N1), A/Hiroshima/52/2005 (H3N2), and B/Malaysia/2506/2004. RE is employed as the toxicity reference in the leukopenic toxicity test (LTT) in Japan [The serum BUN, CRE, CPK, ALT, AST, T-Bil, LDH, ALP, and CRP levels were measured by means of a DRI-CHEM . The SP-D levels were analyzed with the Quantikine Human/Mouse SP-D ELISA Kit . One study indicates that serum SP-D levels increase in response to acute lung inflammation in mice ; therefoThe lungs, liver, and spleen were excised and fixed in 10% neutral-buffered formalin. The fixed samples were dehydrated in graded alcohol washes and embedded in paraffin. Serial sections of 4 μm thickness were stained with hematoxylin and eosin (H&E) and examined under a microscope for routine histopathological examination. Images were acquired by means of an Olympus BX53 microscope .HPRT-1) were quantified simultaneously, and the ratio of each marker gene expression to that of HPRT-1 was calculated.The lung lysates were prepared, and QuantiGene Plex (QGP) was performed to analyze the expression levels of marker genes in lungs as described in our previous studies –6. Detec2+- and Mg2+-free PBS, and then 0.05% Triton X-100 was added to a portion of the homogenized lung lysate, and the sample was incubated for 2 h at 4°C with intermittent mixing. At the end of incubation, the supernatant was collected by centrifugation. Cytokine analyses were performed using the Cytokine 30-Plex Human Panel Kit .For preparation of a lung lysate, a lung was homogenized with CaP < 0.05 were considered statistically significant.For multiple comparisons, one-way analysis of variance followed by Dunnett's multiple-comparison test were performed. The statistical analyses were conducted in GraphPad Prism 6 . Data with